Concentration and cleanup of nucleic acid samples

ABSTRACT

Methods and devices are described for concentration and cleanup of samples containing bio-molecule analytes (e.g., polynucleotides, such as DNA, RNA, PNA). Various embodiments provide for pH-mediated sample concentration and cleanup of nucleic acid samples with channel devices (e.g., cross-T format, microchannel devices).

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a non-provisional of application serial No.60/318,269, filed Sep. 7, 2001, which is incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods and devices for theconcentration and cleanup of samples containing analytes. Aspects of theinvention relate to pH-mediated sample concentration and cleanup ofnucleic acid samples with channel devices.

REFERENCES

[0003] Backhouse et al., DNA sequencing in a monolithic microchanneldevice, Electrophoresis 2000, 21, 150-156.

[0004] Dolnik et al., Capillary electrophoresis on microchip,Electrophoresis 2000, 21, 41-54.

[0005] Grossman and Colburn, Capillary Electrophoresis Theory andPractice, Chapter 1, Academic Press (1992).

[0006] Kambara et al., U.S. Pat. No. 5,192,142 (1993).

[0007] Madabhushi et al., U.S. Pat. No. 5,552,028 (1996).

[0008] Sambrook et al., eds., Molecular Cloning: A Laboratory Manual,Second Edition, Chapter 5, Cold Spring Harbor Laboratory Press (1989).

[0009] Woolley et al., Ultra-high-speed DNA fragment separations usingmicrofabricated capillary array electrophoresis chips, Proc. Natl. Acad.Sci., vol. 91, pp. 11348-11352, Nov. 1994, Biophysics.

[0010] Xiong et al., Base Stacking: pH-Mediated On-Column SampleConcentration for Capillary DNA Sequencing. Anal. Chem. 1998, 70,3605-3611.

BACKGROUND OF THE INVENTION

[0011] In many techniques of molecular biology, it is important to havesamples of high quality. Results are generally enhanced in PCR,sequencing, fragment analysis, and so forth, when the subjectbio-molecule materials are separated from potentially interferingcontaminants. Thus, it is often desirable to purify/concentrate thebio-molecules (e.g., polynucleotides, such as DNA, RNA, PNA, etc.) ofinterest in samples prior to analysis.

[0012] In analyses utilizing laser-induced fluorescence (LIF) detectiontechniques, typical DNA samples often contain, in addition todye-labeled DNA: salts, residual enzyme, DNA oligonucleotides, dNTP's,dye-labeled ddNTP's, and/or surfactants. It is generally desirable toremove all species except the subject dye-labeled DNA fragments.However, even partial purification can be useful. Thus, at a minimum, itis often desirable to remove at least some of the species that arepresent at higher concentration and that could interfere with theanalysis.

[0013] Sample concentration can be used to improve the detection limitsof various analytical methods, such as electrophoresis. For example, thestarting zone length of a sample injection can be reduced by utilizationof a process termed “stacking.” Stacking reduces the width of the samplezone before separation, which can result in improved sensitivity andincreased peak efficiency.

[0014] Xiong et al. describe a method for pH-mediated sampleconcentration of DNA sequencing samples on a capillary tube. While thetechnique of Xiong et al. might allow for sufficient signal from directload on unpurified sequencing samples, it would not be expected toremove unincorporated dyes and contaminants that can obscure thesequencing data. Briefly, according to the method of Xiong et al., acapillary is filled with a nucleic acid DNA separation polymer. However,the polymer solution is buffered with a basic buffer that is charged atlow pH but neutralized at high pH. Xiong et al. employed Tris buffer forthis purpose. The first stage of the process involves a very longelectrokinetic injection from unpurified sequencing reaction (e.g.,right off a thermocycler with no following cleanup step). Because thesample is very salty at this point, the electrokinetic injection processis inefficient and a long injection time is needed to move enough DNAinto the capillary to obtain sufficient signal. The injection time is solong that the peaks would be far too broad to achieve the necessaryresolution for DNA sequencing. To re-focus the DNA starting band, Xionget al. follow the DNA injection with a period of electrophoresis from asodium hydroxide solution. The hydroxide migrates into the capillary,neutralizing the Tris buffer as it enters. In the area where the Tris isneutralized the conductivity becomes very low and therefore the electricfield increases. The increased electric field at the injection end ofthe capillary allows the DNA at the back of the injection plug to travelfaster than the DNA at the front of the injection plug. This refocusesthe injection plug and allows for reasonable resolution to be obtained.When this technique is used with standard capillary electrophoresis, thecontaminating dye labeled terminators, which are present in much higherconcentration than the DNA, also migrate into the separation capillary.The large concentration of dye can migrate with the DNA and maynegatively impact some section of the sequencing data.

SUMMARY OF THE INVENTION

[0015] Aspects of the present invention relate to sample concentrationand cleanup; e.g., cleanup of a DNA sample to reduce or eliminateunincorporated dyes. Among other things, the present invention providesfor direct loading of unpurified sequencing reactions on microfabricatedseparation devices. By way of the present methods and devices, the needto purify sequencing reactions (e.g., after themocycling, whichtypically involves centrifugation) can be reduced or eliminated.

[0016] Aspects of the present invention relate to a channel device,various embodiments of which include (i) an injection channel and aseparation channel, each channel having a first end and a second end,with the separation channel intersecting the injection channel at aregion between the ends of the injection channel; (ii) a first reservoirdisposed for fluid communication with one of the ends of the injectionchannel; (iii) a first separation medium held within the injectionchannel; and (iv) a second separation medium held within the separationchannel; wherein the second separation medium differs from the firstseparation medium;

[0017] Aspects of the present invention relate to a sample-manipulationmethod using such a channel device. For example, various embodiments ofmethods herein include (a) introducing a sample, including apolynucleotide-analyte component and one or more contaminants, into theinjection channel; (b) introducing a pH-modulating composition into theinjection channel; (c) stacking the polynucleotide-analyte component ata stacking region of the device defined by the intersection of thechannels, and locating the one or more contaminants of the sample at aregion between the stacking region and the second end of the injectionchannel; and (d) electrophoresing the polynucleotide-analyte componentdown at least a portion of the separation channel, with a substantialamount (e.g., a majority) of the one or more contaminants remaining inthe injection channel.

[0018] Aspects of the present invention relate to a sample-manipulationmethod, various embodiments of which include (a) providing a channeldevice, the device including (i) an elongate injection channel and anelongate separation channel, each channel having a first end and asecond end, with the separation channel intersecting the injectionchannel at a region between the ends of the injection channel (e.g., ina cross-T format), and (ii) a loading region disposed for fluidcommunication with the first end of the injection channel; (b) placing asample containing a polynucleotide analyte (e.g., DNA) and one or morecontaminants into the loading region; (c) applying a first driving force(e.g., electric field) sufficient to cause at least some of sample tomove from the loading region into the injection channel; (d) placing abasic solution (e.g., NaOH) into the loading region; (e) applying asecond driving force (e.g., electric field) sufficient (i) to cause atleast some of the basic solution to move from the loading region intothe injection channel, thereby causing the polynucleotide analyte tostack in the region of the intersection of the channels, and (ii) tocause at least a portion of the one or more contaminants to move to aregion between the intersection of the channels and the second end ofthe injection channel; and (f) applying a third driving force (e.g.,electric field) sufficient to cause at least a portion of the stackedDNA to move into and along at least a portion of the separation channel,leaving behind in the injection channel at least a substantial portion(e.g., most) of the contaminants.

[0019] According to various embodiments, a separation medium is placedin each of the injection and separation channels. The separation mediumcan be the same in each of the channels, or it can differ in one or morerespects (e.g., concentration and/or composition).

[0020] Aspects of the present invention relate to a sample-manipulationmethod, various embodiments of which include: (a) providing a channeldevice, the device including an injection channel and a separationchannel, each channel having a first end and a second end, with theseparation channel intersecting the injection channel at a regionbetween the ends of the injection channel (e.g., in a cross-T format);(b) introducing a sample, including a polynucleotide-analyte component(e.g., DNA) and one or more contaminants (e.g., unincorporated dyeterminators), into the injection channel; (c) introducing apH-modulating composition (e.g., a basic solution, such as NaOH) intothe injection channel; (d) stacking the polynucleotide-analyte componentat a stacking region of the device defined by the intersection of thechannels, and locating the one or more contaminants of the sample at aregion between the stacking region and the second end of the injectionchannel; and (e) electrophoresing the polynucleotide-analyte componentdown at least a portion of the separation channel, with at least asubstantial portion (e.g., most) of the one or more contaminantsremaining in the injection channel.

[0021] According to various embodiments, a separation medium is placedin each of the injection and the separation channels. The separationmedium placed in the injection channel can differ from, or be the sameas, the separation medium placed in the injection channel. In variousembodiments, the separation medium in the injection channel differs fromthat in the separation channel in one or both of concentration andcomposition.

[0022] In various embodiments, the sample is a DNA sequencing sample(e.g., Sanger sequencing reaction).

[0023] Aspects of the present invention relate to a sample-manipulationmethod, various embodiments of which include: (a) providing a channeldevice, the device including (i) a first channel (e.g., an injectionchannel) and a second channel (e.g., a separation channel), each channelhaving a first end and a second end, with the second channelintersecting the first channel at a region between the ends of the firstchannel (e.g., in a cross-T format); (ii) a loading region (e.g.,reservoir or well) disposed for fluid communication with one of the endsof the first channel; (iii) a separation medium (e.g., polymer-buffercomposition) held within the first and second channels; (b) introducinga sample, including a polynucleotide-analyte component (e.g., DNA) andone or more contaminants (e.g., unincorporated dye terminators), intothe channel by way of the loading region; (c) stacking thepolynucleotide-analyte component at a stacking region of the devicedefined by the intersection of the channels, and locating the one ormore contaminants of the sample at a region between the stacking regionand the second end of the first channel; (d) electrophoresing thepolynucleotide-analyte component down at least a portion of the secondchannel, with a substantial amount (e.g., at least most) of the one ormore contaminants remaining in the first channel; and (e) detecting forthe polynucleotide-analyte component.

[0024] Aspects of the present invention related to a channel device.According to various embodiments, the device includes: (a) an injectionchannel and a separation channel, each channel having a first end and asecond end, with the separation channel intersecting the injectionchannel at a region between the ends of the injection channel; (b) afirst (e.g., loading) reservoir disposed for fluid communication withone of the ends of the injection channel; (c) a first separation mediumheld within the injection channel; and (d) a second separation mediumheld within the separation channel; wherein the second separation mediumdiffers from the first separation medium (e.g., in concentration and/orcomposition).

[0025] According to various embodiments, the injection and theseparation channels are disposed in a cross-T format.

[0026] According to various embodiments, the device further includes asecond reservoir disposed for fluid communication with the other of theends of the injection channel.

[0027] According to various embodiments, the device further includesthird and fourth reservoirs, each being disposed for fluid communicationwith a respective one of the first and second ends of the separationchannel.

[0028] According to various embodiments, each of the first and secondseparation media includes a polymer component, with the polymercomponent in the injection channel being present at a higherconcentration than the polymer component in the separation channel.

[0029] According to various embodiments, the device further includeseach of the first and second separation media includes a buffercomponent. According to various embodiments, the buffer componentsdiffer from one another. The buffer component of the first separationmedium can comprise, for example, a Tris-HCl buffer. The buffercomponent of the second separation medium can comprise, for example, aTAPS/Tris buffer.

[0030] According to various embodiments, one of the ends of theseparation channel intersects (for fluid communication with) theinjection channel.

[0031] According to various embodiments, a region between the ends ofthe separation channel intersects the injection channel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The structure and manner of operation of the invention mayfurther be understood by reference to the following description taken inconjunction with the accompanying drawings, in which identical referencenumerals identify identical or similar elements, and in which:

[0033]FIG. 1 is a partially schematic perspective view from one side ofa microfabricated channel device, useful in practicing the presentinvention; and

[0034]FIGS. 2A, 2B, 2C and 2D schematically depict an example of thepresent invention. Unpurified DNA is provided in a loading reservoir ofa cross-T format channel device (FIG. 2A). The unpurified DNA sample isthen caused to flow into and along an injection arm of the device (FIG.2B). DNA is then stacked in a region whereat the injection andseparation channels intersect (FIG. 2C). The concentrated DNA is thenintroduced into a separation arm of the device, leaving behind in theinjection arm various impurities, such as unincorporated dyes (FIG. 2D).

DESCRIPTION OF THE INVENTION

[0035] Reference will now be made to various embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction withvarious preferred embodiments, it will be understood that they are notintended to limit the invention. On the contrary, the invention isintended to cover alternatives, modifications, and equivalents, whichmay be included within the invention as defined by the appended claims.

[0036] Unless stated otherwise, the following terms and phrases as usedherein are intended to have the following meanings:

[0037] The term “channel” as used herein refers to an elongate, narrowpassage or other structure (e.g., grooves, etc.) formed in a substrateand capable of supporting a volume of separation medium and/or buffersolution; e.g., such as is used in carrying out electrophoresis. Thegeometry of a channel may vary widely. For example, a channel can have acircular, oval, semi-circular, semi-oval, triangular, rectangular,square, or other cross-section, or a combination thereof. Channels canbe fabricated by a wide range of technologies, includingmicrofabrication techniques. As used herein, the term “channel” is notintended to encompass a capillary tube.

[0038] The terms “capillary” and “capillary tube” as used herein, referto an elongated tubular or cylindrical structure defining an innerlumen. For example, a capillary can be an elongated capillary ormicro-capillary tube made, for example, from fused silica, quartz,silicate-based glass, such as borosilicate glass, phosphate glass,alumina-containing glass, and the like, or other silica-likematerial(s). As used herein, “capillary” does not encompass a channel ina substrate such as a plate, slide, chip, wafer, or the like.

[0039] The terms “channel device” and “microchannel device” refer to asubstrate, such as a plate, slide, chip, wafer, or similar structure,including one or more channels (e.g., grooves); and particularly thoseadapted at least in part for carrying out electrophoresis. Channeldevices can take the form, for example, of microfabricated devices(e.g., a grooved, etched, or fluted plate, slide, chip, wafer, or othersubstrate).

[0040] The terms “concentrate,” “purify,” and “cleanup” refer to theremoval or separation of a substance or material from an original, orstarting, state or environment. For example, a material is said to be“purified” when it is present in a particular composition in a higherconcentration than exists as it is found in a starting sample. Forexample, where a starting sample comprises a polynucleotide in a crudecell lysate, the polynucleotide can be said not to be purified, but thesame polynucleotide separated from some or all of the coexistingmaterials in the cell lysate is purified. In another example, where astarting sample comprises analyte DNA and one or more contaminants, suchas unincorporated dyes, salts, residual enzyme, undesired DNAoligonucleotides, dNTP's, dye-labeled ddNTP's, and/or surfactants, theanalyte DNA can be said not to be “cleaned up,” but the same analyte DNAseparated from some or all of the contaminant(s) is said to be “cleanedup.”

[0041] As used herein, the terms “separation medium” and “separationmatrix” refer to a medium in which an electrophoretic separation ofsample components can take place. Separation media typically compriseseveral components, at least one of which is a charge-carryingcomponent, or electrolyte. The charge-carrying component is usually partof a buffer system for maintaining the separation medium at a definedpH. Media for separating polynucleotides, proteins, or otherbiomolecules having different sizes but identical charge-frictional dragratios in free solution, further include a sieving component. Suchsieving component is typically composed of a cross-linked polymer gel,e.g., crosslinked polyacrylamide or agarose (Sambrook), or a polymersolution, e.g., a solution of polyacrylamide, hydroxyethyl cellulose,and the like (Grossman; Madabhushi).

[0042] In various embodiments, separation channels are formed in a glassor plastic substrate, such as a plate, slide, wafer, chip, or the like,by fabrication techniques known in the art, e.g., photolithographicaland/or wet-chemical etching procedures, laser ablation, electroforming,microcontact printing, microstamping, micromolding, microcasting,micromachining, engraving, and/or embossing techniques, to name a few.For example, Backhouse et al., Dolnik et al., and Woolley et al (each ofwhich is incorporated herein by reference) discuss certainmicrofabrication techniques that the skilled artisan can employ inmaking the devices of the present invention. In one embodiment, theseparation channels are formed in a generally planar substrate comprisedat least in part, for example, of an electrically insulating material,e.g., fused silica, quartz, silicate-based glass, such as borosilicateglass, phosphate glass, alumina-containing glass, and the like, or othersilica-like material(s).

[0043] According to various embodiments of the present invention, a pHmediated stacking process is used in the injection arm of a cross-Tinjection arrangement, such as can be formed in a microfabricatedelectrophoresis device. Contaminants, such as contaminating dyeterminators, migrate out into a waste region or reservoir and, thus, arenot injected into an associated separation arm with the bio-moleculeanalyte(s).

[0044] A microfabricated device can be particularly advantageous becausedifferent polymers and/or buffers may be utilized in the injection armand the separation channel. For example, one polymer or polymer-buffercombination can be held in the injection channel, while another(different) polymer or polymer-buffer combination can be held in theseparation channel. According to various embodiments, high concentrationpolymer and a buffer, such as Tris-HCl, is held in the injection arm ofa channel device, while low concentration polymer and a buffer, such asTAPS/Tris, is held in the separation channel.

[0045] According to various embodiments, both an injection channel and aseparation channel of a cross-T channel device hold identical polymersand/or buffers. As indicated above, according to other embodiments, oneor both of the buffer and polymer differ in the separation channel andthe injection channel.

[0046] In certain embodiments, the buffer and/or polymer is of a higherconcentration in one of the channels as compared to that held in theother of the channels. For example, a high concentration buffer can beheld in one channel (e.g., about 100 mM or higher) and a lowconcentration buffer (e.g., 25 mM and below) can be held in the otherchannel. In an embodiment, the injection channel holds a highconcentration buffer while the separation channel holds a lowconcentration buffer. In various embodiments, a high concentrationpolymer can be held in one channel (e.g., 5% polyacrylamide or PDMA, orhigher) and a low concentration polymer (e.g., less than 5%polyacrylamide or PDMA) can be held in the other channel.

[0047]FIG. 1 depicts general features of one type of device in which thepresent invention can be embodied. It will be appreciated that otherconfigurations may be employed. The channel device of FIG. 1, indicatedgenerally by the reference numeral 10, comprises a substrate 12 in whichchannels, such as 14 and 16, are defined so as to intersect at rightangles at a junction, denoted at 18. More particularly, substrate 12 iscomprised of lower and upper plates, 20 and 22 respectively, withabutted confronting faces. Lower plate 20 is provided with elongategrooves, each of roughly semi-circular or semi-oval cross-section, thatin part define boundaries for channels 14, 16. The lower face of plate22 is substantially planar, and, when disposed against plate 20 asshown, further defines boundaries for channels 14, 16. Particularly, inthe illustrated arrangement, the grooves of plate 20 define lower(floor) and side walls or boundaries of each channel 14, 16 and thelower surface of plate 22 provides an upper wall or ceiling (boundary)for channels 14, 16.

[0048] In various embodiments, discussed further herein, channel 14 canbe employed as an “injection channel” and channel 16 can be employed asa “separation channel.”

[0049] Several electrodes are provided, schematically indicated as 24,26, 28 and 30; each being disposed for electrical communication with areservoir, such as 34, 36, 38 and 40, respectively. One or more powersources (not shown) are disposed for electrical communication with theelectrodes, permitting selective establishment of defined DC fieldsalong the channels. For example, one DC field may be established alongthe injection channel. Another DC field may be established along theseparation channel. The fields can be established one at a time, orsimultaneously, as desired.

[0050] Reservoirs 34, 26, 38, 40 are defined by small through-holes;drilled, etched, punched, or otherwise formed through upper plate 22.Each of reservoirs 34, 36, 38, 40 is disposed for fluid communicationwith a respective end of one of channels 16, 18, as shown.

[0051] For reasons that will become apparent, it is convenient to referto channels 14, 16 as comprising four segments or arms, denoted as 1, 2,3 and 4 in FIG. 1. More particularly, segments 1, 2, and 3 are referredto herein as “side arms,” or “injection arms;” and segment 4 is referredto herein as a “separation arm” or “main arm.”

[0052] The channels can be any suitable length, and any suitableprofile. In one exemplary configuration, main arm 4 is 50 micrometerswide (measured at its top, from one lateral side wall to an opposinglateral side wall) and 20 micrometers deep (measured from its upperceiling or top wall to a lowermost region of its bottom wall or floor),with a length of 8.5 centimeters. The side arms can also be any suitablegeometry, including non-straight geometries, and any suitable length. Inthis embodiment, each of side arms 1, 2, 3 has the same cross-sectionalprofile (width and depth) as the long channel, and a length of 1centimeter. One suitable channel device for use in the presentinvention, having such dimensions, is the Standard Microfluidic Chip(Simple Cross, MC-BF4-SC) from Micralyne Inc. (Edmonton, Alberta,Canada). Multiple cross-channel or other channel arrangements can beprovided on a single chip or plate, as desired. A cross-channelconfiguration, such as depicted in FIG. 1, is often referred to in theart as a “T” format (the “T” representing the intersection of thechannels).

[0053] It should be appreciated that the present invention is notlimited to the construction depicted in FIG. 1, but rather many deviceconfigurations are possible and can be used in the context of thepresent invention. For example, while only one T-format cross-channelarrangement is shown in FIG. 1, any reasonable number of sucharrangements can be provided on a substrate. In one embodiment, both theupper and lower plates are provided with complimentary grooveconfigurations that are aligned with one another so that correspondingupper and lower grooves cooperate to define one or more channels. Inanother embodiment, a plurality of spacer strips are placed betweenplanar, parallel, opposed surfaces of confronting plates. The spacerstrips, in this embodiment, define the distance separating the opposedplate surfaces, and the region between adjacent pairs of spacersdefines, at least in part, each of one or more channels. Particularly,one or both of the lateral sides of each spacer define channel sideboundaries and the planar confronting plate surfaces define upper andlower boundaries.

[0054] Instead of providing grooves in a lower plate that are covered byan upper plate, such as shown in FIG. 1, a channel device can include anupper plate with grooves formed along its lower surface, which can beplaced over a planar upper surface of a lower plate. Moreover, althoughthe channel device shown in FIG. 1 is disposed with its major planarsurfaces disposed in a substantially horizontal fashion, the devicecould instead be disposed with its major planar surfaces disposedsubstantially vertically, or tilted at a desired angle. These and othervariations and adaptations can readily be selected and implemented bythe skilled artisan.

[0055] Other features that can be included in a channel device for useherein can be found, for example, in the References hereto (each ofwhich is incorporated herein by reference).

[0056] In practice, a separation medium can be injected (e.g.,pressure-filled or vacuum aspirated) or otherwise provided in thechannels of the device. Exemplary separation mediums include but are notlimited to agarose and crosslinked polyacrylamide. In one embodiment,GeneScan Polymer (P/N 401885) and/or POP-6 (P/N 402844) from AppliedBiosystems (Foster City, Calif.) are employed as a separation medium.

[0057] In one embodiment, a sample containing a polarizable analyte andone or more contaminants is placed in one of reservoirs, 34, 36, 38, 40;and buffer solution is placed in one or more of the other reservoirs.Loading can be effected in any suitable manner, e.g., by way of a manualor automated pipette assembly.

[0058] Various embodiments of the present invention make use of across-channel or T-format geometry and DC electric fields toconcentrate/purify DNA away from potentially interfering species in abulk solution. Selectively applied DC fields and a pH adjustment withinthe injection channel results in DNA concentration into a small volumewithin the microfabricated device, e.g., at the intersection of thechannels. Once concentrated into a small volume and purified away fromat least some, and preferably most, of the potential interferences inthe bulk solution, the DNA can be moved into a separation channel orcollection reservoir for analysis and/or recovery.

[0059] Various embodiments are particularly adapted to bio-molecule(e.g., DNA, RNA, PNA, etc.) sequence or other analysis methods, in whicheach of a plurality of different fragment types is labeled with aspectrally distinctive fluorescent dye. According to certainembodiments, a laser is adapted to direct an excitation beam of coherentlight at a detection zone, at a location along or a separation channel,or just beyond an outlet end of such channel, of a channel device. Theexcitation beam excites the dyes to emit light. In various embodiments,emitted light from sample zones passes through a collection lens,through a laser light filter, and through a focusing lens. The focusedlight can be incident on a detector array (e.g., CCD) capable ofdetecting the emissions from the detection zone of the channel.Electronic signals from the detector array can provide information aboutthe character or sequence of the bio-molecule sample.

[0060] According to various embodiments, DNA fragments of interest areconcentrated and purified away from interfering species and injectedinto an analyzer with little or no user intervention or manipulation. Incertain of the embodiments herein, DNA concentration and purification isintegral with a separation device and, thus, requires no transfer ofsamples from the purification device to the analyzer. One suchembodiment will now be described in the context of Example 1.

[0061] It is to be noted that the following example is merelyillustrative, and not limiting, of the present invention.

EXAMPLE

[0062] This non-limiting example illustrates use of the present methodsand devices in sample cleanup and injection; e.g., cleanup and injectionof a DNA-containing sample prior to electrophoretic analysis. As thisexample illustrates, the methods and devices herein can be used, forexample, to inject a concentrated plug of DNA into a channel,substantially free from salts and dye terminators that are in the bulksolution.

[0063] Reference is now made to FIGS. 2A, 2B, 2C and 2D, whichschematically depict the present example.

[0064] A cross-T format channel device 10 is provided, including (i) ahigh concentration polymer and Tris-HCl buffer in an injection channel14 and (ii) a low concentration sieving polymer and TAPS/Tris buffer ina separation channel 16. An unpurified DNA sample 52 is loaded in areservoir 36 of the channel device 10 (FIG. 2A).

[0065] The unpurified DNA sample is then caused to flow into and alongan injection arm of the device (FIG. 2B). For example, under theinfluence of a DC potential, the DNA-containing sample can beelectrophoresed to introduce it into the device, such that DNA andpotentially interfering components, such as unincorporated dyes, becomedistributed within injection channel 14. For example, a potential of100V DC can be applied between reservoirs 36 and 40, therebyelectrophoretically pulling DNA-containing sample into injection channel14. Reservoir 36, which had held sample 52, is then loaded with NaOH(FIG. 2B).

[0066] DNA is then stacked in a region whereat the injection andseparation channels intersect (note the stacked DNA, indicated at 54 inFIG. 2C). This can be accomplished by electrophoresing the NaOH intoinjection channel 14, thereby causing the DNA to “stack up,” whilecontaminants, such as dyes 56, migrate ahead (i.e., further downstreamalong the injection channel than the DNA).

[0067] At least a portion of the stacked DNA 54 is then introduced intoseparation channel 16 of device 10 (FIG. 2D). For example, the DNA canbe introduced into separation channel 16 by discontinuing the injectionvoltage along the injection channel and applying a separation voltage(1000V DC) down the length of the separation channel (see FIG. 2D). Oncein the channel, the injected DNA (denoted as 54 a in FIG. 2D) can beresolved. The resolved DNA can be detected (e.g., using a LIF detectionarrangement disposed to observe a downstream region along the separationarm) and/or recovered.

[0068] All publications and patent applications referred to herein arehereby incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0069] Those having ordinary skill in the electrophoresis art willclearly understand that many modifications are possible in the abovepreferred embodiments without departing from the teachings thereof. Allsuch modifications are intended to be encompassed within the followingclaims.

It is claimed:
 1. A sample-manipulation method, comprising: (a)providing a channel device, said device including (i) an injectionchannel and a separation channel, each channel having a first end and asecond end, with said separation channel intersecting the injectionchannel at a region between the ends of the injection channel; (ii) afirst reservoir disposed for fluid communication with one of said endsof said injection channel; (iii) a first separation medium held withinsaid injection channel; and (iv) a second separation medium held withinsaid separation channel; wherein said second separation medium differsfrom said first separation medium; (b) introducing a sample, including apolynucleotide-analyte component and one or more contaminants, into saidinjection channel; (c) introducing a pH-modulating composition into saidinjection channel; (d) stacking said polynucleotide-analyte component ata stacking region of said device defined by the intersection of thechannels, and locating said one or more contaminants of said sample at aregion between the stacking region and said second end of said injectionchannel; and (e) electrophoresing said polynucleotide-analyte componentdown at least a portion of said separation channel, with at least mostof said one or more contaminants remaining in said injection channel. 2.A sample-manipulation method, comprising: (a) providing a channeldevice, said device including (i) an elongate injection channel and anelongate separation channel, each channel having a first end and asecond end, with said separation channel intersecting the injectionchannel at a region between the ends of the injection channel, and (ii)a loading region disposed for fluid communication with said first end ofsaid injection channel; (b) placing a sample containing a polynucleotideanalyte and one or more contaminants into said loading region; (c)applying a first driving force sufficient to cause said sample to movefrom said loading region into the injection channel; (d) placing a basicsolution into said loading region; (e) applying a second driving forcesufficient (i) to cause said basic solution to move from said loadingregion into the injection channel, thereby causing the polynucleotideanalyte to stack in the region of the intersection of the channels, and(ii) to cause at least a portion of the one or more contaminants to moveto a region between the intersection of the channels and said second endof said injection channel; and (f) applying a third driving forcesufficient to cause at least a portion of the stacked DNA to move intoand along at least a portion of the separation channel, leaving behindin the injection channel at least most of the contaminants.
 3. Themethod of claim 2, wherein said injection and separation channels arearranged in a cross-T format.
 4. The method of claim 2, wherein each ofsaid first, second and third driving forces comprises an electric field.5. The method of claim 2, wherein said basic solution comprises NaOH. 6.The method of claim 2, wherein said polynucleotide analyte comprisesDNA.
 7. The method of claim 2, further comprising placing a separationmedium in each of said injection and separation channels.
 8. The methodof claim 7, wherein the separation medium placed in said injectionchannel differs from the separation medium placed in the injectionchannel.
 9. The method of claim 8, wherein the separation media differin one or both of concentration and composition.
 10. Asample-manipulation method, comprising: (a) providing a channel device,said device including an injection channel and a separation channel,each channel having a first end and a second end, with said separationchannel intersecting the injection channel at a region between the endsof the injection channel; (b) introducing a sample, including apolynucleotide-analyte component and one or more contaminants, into saidinjection channel; (c) introducing a pH-modulating composition into saidinjection channel; (d) stacking said polynucleotide-analyte component ata stacking region of said device defined by the intersection of thechannels, and locating said one or more contaminants of said sample at aregion between the stacking region and said second end of said injectionchannel; (e) electrophoresing said polynucleotide-analyte component downat least a portion of said separation channel, with at least most ofsaid one or more contaminants remaining in said injection channel. 11.The method of claim 10, wherein said injection and separation channelsare arranged in a cross-T format.
 12. The method of claim 10, whereinsaid pH-modulating composition comprises a basic solution.
 13. Themethod of claim 12, wherein said basic solution comprises NaOH.
 14. Themethod of claim 10, wherein said polynucleotide-analyte componentcomprises DNA.
 15. The method of claim 10, further comprising placing aseparation medium in each of said injection and separation channels. 16.The method of claim 15, wherein the separation medium placed in saidinjection channel differs from the separation medium placed in theinjection channel.
 17. The method of claim 16, wherein the separationmedia differ in one or both of concentration and composition.
 18. Themethod of claim 10, wherein said one or more contaminants include one ormore dyes.
 19. The method of claim 10, wherein said sample is a DNAsequencing sample.
 20. A sample-manipulation method, comprising: (a)providing a channel device, said device including (i) a first channeland a second channel, each channel having a first end and a second end,with said second channel intersecting the first channel at a regionbetween the ends of the first channel; (ii) a loading region disposedfor fluid communication with one of said ends of said first channel;(iii) a separation medium held within said first and second channels;(b) introducing a sample, including a polynucleotide-analyte componentand one or more contaminants, into said first channel by way of saidloading region; (c) stacking said polynucleotide-analyte component at astacking region of said device defined by the intersection of thechannels, and locating said one or more contaminants of said sample at aregion between the stacking region and said second end of said firstchannel; (d) electrophoresing said polynucleotide-analyte component downat least a portion of said second channel, with at least most of saidone or more contaminants remaining in said first channel; and (e)detecting for said polynucleotide-analyte component.
 21. A channeldevice, comprising: an injection channel and a separation channel, eachchannel having a first end and a second end, with said separationchannel intersecting the injection channel at a region between the endsof the injection channel; a first reservoir disposed for fluidcommunication with one of said ends of said injection channel; a firstseparation medium held within said injection channel; and a secondseparation medium held within said separation channel; wherein saidsecond separation medium differs from said first separation medium. 22.The device of claim 21, wherein said injection and separation channelsare disposed in a cross-T format.
 23. The device of claim 21, furthercomprising a second reservoir disposed for fluid communication with theother of said ends of said injection channel.
 24. The device of claim23, further comprising third and fourth reservoirs, each being disposedfor fluid communication with a respective one of said first and secondends of said separation channel.
 25. The device of claim 21, whereineach of said first and second separation media includes a polymercomponent, with the polymer component in said injection channel beingpresent at a higher concentration than the polymer component in saidseparation channel.
 26. The device of claim 21, wherein each of saidfirst and second separation media includes a buffer component.
 27. Thedevice of claim 26, wherein said buffer components differ from oneanother.
 28. The device of claim 26, wherein the buffer component ofsaid first separation medium comprises a Tris-HCl buffer.
 29. The deviceof claim 26, wherein the buffer component of said second separationmedium comprises a TAPS/Tris buffer.
 30. The device of claim 21, whereinone of said ends of said separation channel intersect the injectionchannel.
 31. The device of claim 21, wherein a region between said endsof said separation channel intersects the injection channel.